A specimen illuminated by fluorescent light on microscope stage in St Johnston lab

Implementation of a two-mirror scanning system enables fast volumetric imaging with a tilted light-sheet produced by a single objective on a fully functional commercial microscope base

14.03.24 Building a user-friendly oblique plane microscope on a recycled commercial microscope base.

A study from the St Johnston lab led by George Sirinakis and Edward Allgeyer used a two-mirror scanning system which enabled them to compensate for changes in the position of the objective lens during routine microscope operation. Compared to the more traditional scan-lens/galvo-mirror combination, the two-mirror scanning geometry offers higher light-efficiency and a more compact footprint, which could be beneficial to all single objective light-sheet microscope designs regardless of the use of a commercial base or not.

Sirinakis G et al, (2024) User-friendly oblique plane microscopy on a fully functional commercially available microscope base. Biomed. Opt. Express 15, 2358-2376. DOI: 10.1364/BOE.518856

User-friendly oblique plane microscopy on a fully functional commercially available microscope base - figure 1

Fig. 1: (a) System layout. OBJ 1-3: objective lenses, TL 1-3: tube lenses, G1-2: galvanometric mirrors, DM: dichroic mirror, M1: mirror, CY 1-3: cylindrical lenses, AP 1-2: adjustable slits, RM: resonance mirror, TS: translation stage. (b) The tertiary objective, tube lens, and camera are placed on a straight path for initial remote focusing characterization, setup, and diagnostic purposes. (c) Top row, Zemax schematic illustrating that galvo’s G1 and G2 can support a ± 10 mm scan of a 20 mm diameter image across the intermediate image plane of the microscope base without clipping. Bottom row, Zemax footprint diagram showing where rays land on the mirror surface. For a 100X magnification, this translates to an area in the sample space of 200 μm in diameter over a scan range of ± 100 μm which extends beyond the supported FOV of the primary objective (OBJ 1).

Abstract from the paper

In this work we present an oblique plane microscope designed to work seamlessly with a commercially available microscope base. To support all the functionality offered by the microscope base, where the position of the objective lens is not fixed, we adopted a two-mirror scanning geometry that can compensate for changes to the position of the objective lens during routine microscope operation. We showed that within a ± 1 mm displacement range of the 100X, 1.35 NA objective lens away from its designed position, the PSF size increased by <3% and <11% in the lateral and axial dimensions, respectively, while the error in magnification was <0.5% within volumes extending ± 10 µm about the focal plane. Compared to the more traditional scan-lens/galvo-mirror combination, the two-mirror scanning geometry offers higher light efficiency and a more compact footprint, which could be beneficial to all OPM designs regardless of the use of a commercial base or not.